Free radical pathways of prostaglandin synthase are being pursued in collaboration with Dr. Eling. Electron spin resonance investigations of the prostaglandin hydroperoxidase and a model enzyme system, horseradish peroxidase, have demonstrated the enzymatic formation of the sulfur trioxide anion (SO(3) from (bi)-sulfite. Prostaglandin synthase forms prostaglandins from arachidonic acid only in the presence of oxygen where direct ESR cannot detect SO(3). This species reacts with the radical trap 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) as evidenced by ESR studies and the complete inhibition of (bi)sulfite-dependent oxygen uptake by DMPO. The metabolism of benzidine and several derivatives was studied using horse-radish peroxidase and prostaglandin synthase. Benzidine was oxidized to a radical cation and a charge-transfer complex composed of the benzidine and its two electron (di-imine) oxidation product. The two-electron oxidation product, the di-imine, is a resonance structure of the nitrenium ion, the proposed ultimate carcinogenic metabolite of aromatic amines. In collaboration with Prof. West fast-flow techniques are being used to detect the highly reactive phenoxyl free radical metabolites of acetaminophen and diethylstilbestrol.